This invention claims priority of the German patent application 100 43 504.1, filed Sep. 1, 2000, which is incorporated by reference herein.
The invention concerns a method and an apparatus for laser microdissection of specimen regions of interest of a specimen that is mounted on a specimen holder.
xe2x80x9cLaser microdissectionxe2x80x9d refers, in the field of biology and medicine, to a method with which a small piece is cut out from a generally flat specimen (for example cells or a tissue section) with a fine, focused laser beam. The cut-out piece is thus available for further biological or medical (e.g. histological) examinations.
U.S. Pat. No. 5,998,129 describes a method of this kind and an apparatus for laser microdissection. The specimen is arranged on a solid, planar support, for example a polymer support film, that is stretched over a glass specimen slide commonly used in laboratories. The method described operates in two steps. In a first step, a specimen region of interestxe2x80x94on which, for example, a selected cell grouping or a histological section is locatedxe2x80x94is cut out with a laser beam. For that purpose, the cut line of the laser beam describes a complete curve around the specimen region of interest. After cutting, the cut-out specimen region of interest is then still adhering to or resting on its substrate. In a second step, an additional laser shot is therefore directed onto the specimen region of interest, and the specimen region of interest is thereby catapulted in the direction of the laser beam into a collection vessel.
One disadvantage of the method occurs already in the first method step. Shortly before the cut line is completed, the cut-out specimen region of interest is joined to the surrounding specimen only by a narrow web. As a result of electrical charging or mechanical stress in the web, at this stage of the cut the previously cut-out specimen region of interest often swings away, i.e. out of the focal plane of the laser beam or behind the remaining support film. It is not possible to complete cutting of the swung-away specimen region of interest, since the folded-over portion of the sample region projects into the cut line and will thus be damaged upon further cutting. At the same time, transporting the cut-out specimen region away by means of a laser shot thereby becomes difficult or even impossible, since a sufficient application area for the laser shot is not present.
It is therefore the object of the invention to describe a method for laser microdissection which allows controlled detachment of a specimen region of interest from a specimen, and prevents the specimen region of interest from swinging away.
This object is achieved by a method for laser microdissection of specimen regions of interest of a specimen that is mounted on a specimen holder which according to the present invention comprises the following steps:
a) creating a perforation with webs along a cut line enclosing the specimen region of interest by means of a focused laser beam, the perforation having at least two webs that interrupt the cut line and join the specimen region of interest to the surrounding specimen; and
b) breaking the webs of the perforation with a single laser pulse of the defocused laser beam directed onto the specimen region of interest, thereby detaching the specimen region of interest from the specimen.
It is a further object of the invention to describe an apparatus for performing the method for laser microdissection which allows controlled detachment of a specimen region of interest from a specimen, and prevents the specimen region of interest from swinging away.
This object is achieved by an apparatus for laser cutting of microscopic specimens with a focused laser beam, which according to the present invention has the following features:
a) a microscope having at least one objective that defines an optical axis, for viewing of a specimen having a specimen region of interest, and having a laser that generates a laser beam and at least one optical system that couples the laser beam into the objective,
b) a cut line control unit, associated with said microscope for generating a relative movement between said laser beam focused by the objective,
c) perforation creating means for creating a perforated cut line around said specimen region of interest, said cut line having at least two webs that join said specimen region of interest to said surrounding specimen,
d) means for defocusing said laser beam, and
e) web breaking means for directing a single laser pulse of said defocused laser beam onto the specimen region of interest, thereby detaching the specimen region of interest from the specimen.
For the method according to the present invention, the specimens to be examined, from which specimen regions of interest are to be cut out, are prepared on very thin plastic films. The thickness of these plastic films is on the order of between 1 and 2 xcexcm. PET films, for example, can be used. The best cutting results have been obtained, however, with PEN films. With these it is possible to generate narrow and at the same time stable webs. It has proven particularly favorable for the method if webs having a width of approximately 1 xcexcm are left in place. The plastic films are stretched, in known fashion, over a specimen holder. This can be, for example, a glass specimen slide commonly used in laboratories. Other specimen holders (in terms of shape and material) are, however, conceivable. The specimen holder rests on an X-Y stage which allows different specimen regions to be viewed and selected. The apparatus usually has at least one vessel in the vicinity of the specimen for collecting a cut-out specimen region of interest.
One embodiment of the apparatus according to the present invention has a stationary laser beam, and the cut line control unit comprises a displaceable X-Y stage which moves the specimen relative to the stationary laser beam during creation of a perforation. In this context, very high demands are made on the positioning accuracy of the X-Y stage in order to produce an exact cut line. The X-Y stage is preferably displaced in motorized fashion.
In another embodiment of the apparatus according to the present invention, the cut line control unit comprises a laser scanning device which moves the laser beam relative to a stationary specimen during creation of a perforation. For that purpose, the X-Y stage with the specimen holder and specimen on it is not displaced during creation of the perforation. The cut line of the perforation results exclusively from deflection of the laser beam over the specimen.
A particularly advantageous embodiment of the apparatus is one in which the means for creating a perforation comprise a laser control unit which controls the operating parameters of the laser. Those operating parameters are, for example, the laser power, the laser pulse duration, or the laser cut width. The means for creating a perforation can additionally comprise an autofocus apparatus for the laser, thereby making possible automation of the perforation operation.
In addition, the means for breaking the webs of the perforation can comprise a perforation control unit for controlling the cut line control unit and the laser control unit. It proves advantageous for this purpose if the perforation control unit additionally comprises means for defocusing the laser. A user of the apparatus for breaking the webs then no longer needs to defocus the laser manually. This defocusing constitutes an essential part of the method, since thereby the laser beam is spread out and its energy is distributed over a larger area. As a result, a cut can no longer be generated in the specimen region of interest, but the laser pulse is used to break the webs.
For laser microdissection of another specimen region of interest, focusing of the laser can then be performed very quickly and reliably by means of the autofocus apparatus. This makes possible automation of the entire method.
In other embodiments of the apparatus, means for selection of the cut line, or means for selection of the cut line and the position of the webs by a user, are provided. In addition, means can be provided for selection of the width of the webs and for selection of the position of the webs by a user. By way of this selection capability, the user can specifically select the correct specimen region of interest before creation of the perforation, and at the same time can protect important portions of the specimen from damage. For example, crack structures occur in the region of the webs between the specimen region of interest and the surrounding specimen. Because the user can, for example, place the webs on non-critical cell structures of the specimen, critical cell structures of interest within the specimen region of interest can be protected from such crack structures.
The method according to the present invention possesses the advantage of ruling out any swinging away of the specimen region of interest during cutting. Problem-free cutting of the specimen is thereby possible. In addition, reliable removal of the cut-out specimen region of interest is possible. Automation of the method and of the apparatus makes possible utilization in routine laboratory operations.